8-pyridyl purines

ABSTRACT

8-PYRIDLY PURINES CAN BE SYNTHESIZED BY CONDENSATION OF 4,5-DIAMINOPYRIMIDINES WITH AMIDINOPYRIDINES. PROTONATION OF THE PYRIDINE NITROGEN CAUSES A MARKED BATHOCHROMIC SHIFT OF $MAX IN THE 2- AND 4-PYRIDYL DERIVATIVES, BUT NOT IN THE 3-PYRIDYL ISOMERS. A SIMILAR EFFECT IS PRODUCED BY QUATERNISATION OF THE 4-, BUT NOT OF THE 3-PRIDYL SUBSTITUENT. THE 8-(N-METHYLPYRIDINIUM) GROUP ALSO FACILITATES GREATLY ANION FORMATION IN THE PRUINE RING. THE NEW COMPOUNDS SHOWED A VARIETY OF BIOLOGICAL ACTIVITIES, SUCH AS PROLONGED HYPOTENSION WHEN INJECTED INTO CATS WHICH WERE ANESTHETIZED WITH NEMBUTAL.

United States Patent Olhc 3,573,309 Patented Mar. 30, 1971 3,573,309S-PYRIDYL PURINES Felix Gotthilf Bergmann, Jerusalem, Mordechai AbrahamKleiner, Petah-Tikva, and Moshe Rashi, Jerusalem, Israel, assignors toYissurn Research Development Company, Jerusalem, Israel No Drawing.Filed July 30, 1968, Ser. No. 748,620 Claims priority, application6Israel, Aug. 4, 1967,

Int. (:1. 607a 57/62 US. Cl. 260--252 14 Claims ABSTRACT OF THEDISCLOSURE 8-pyridyl purines can be synthesized by condensation of4,5-dia-minopyrimidines with amidinopyridines. Protonation of thepyridine nitrogen causes a marked bathochromic shift of m in the 2- and4-pyridyl derivatives, but

not in the 3-pyridy1 isomers. A similar effect is produced byquaternisation of the 4-, but not of the 3-pyridy1 substituent. TheS-(N-methylpyridinium) group also facilitates greatly anion formationinthe purine ring.

The new compounds vshowed a ,variety of biological activities, such asprolonged hypotension when injected into cats which were anesthetizedwith nembutal.

which are useful for enzymological and chemotherapeutic studies. Thesecompounds have the sameuses as analogous known compounds and, inaddition, certainof these compounds exhibit brilliant yellow oryellow-green fluorescence. The compounds produced in accordance with thepresent invention include'hypoxanthines, Xanthines, 6- thioxanthines,G-mercaptopurines and S-pyridyl derivatives of purine itself.

Among the compounds of the present invention are 8-(2-pyridyl) purine;

8-(3'-pyridyl) purine;

8- (4'-pyridyl) purine; 8-(2-pyridyl)-6-rnercaptopurine;8-(3'-pyridyl)-6-mercaptopurine;

8- (4-pyridyl -6-mercaptopurine;

8- (2'-pyridyl) -6-thioxanthine;

8- 3'-pyridyl) -6-thioxanthine;

8- 4'-pyridyl) -6-thioxanthine;

8-(p-anisyl) hypoxanthine;

8- (p-nitrophenyl) hypoxanthine; 8-(2'-pyridyl) hypoxanthine;

8-(3'-pyridyl) hypoxanthine;

8-(4'-pyridyl) hypoxanthine;

8-(2'-pyridy1) xanthine;

8-(3-pyridyl) xanthine;

8-(4'-pyridyl) xanthine;

8-(3-pyridyl) hypoxanthine-1-methiodide; 8-(4-N-methylpyridinium)hypoxanthine betaine; 8-(3'-N-methylpyridinium) Xanthine betaine; 8-(4'-N-methylpyridinium) xanthine betaine; 8- 2-pyridyl -6-methy1thiopurine; 8-(1-methyl pyridiniurn) purines iodides;8-(1'-methylpyridinium) purines betaines;

8- 2-pyridyl) -3-methyl hypoxanthine;

8- 4'-N-methylpyridinium) -3 -methyl-6-methylthio purine iodide;

8- (4'-N-methylpyridinium) -3 -methyl-6-thio purine betaine;

8- (4'-pyridyl)-1-methyl-hypoxanthine;

8- (4'-pyridyl)-1-methyl-6-thiopurine;

8- 4-pyridyl l -methyl-6-methylthiopurine;

8- (4'-N-'methylpyridinium) -1-methyl-6-methylthiopurine iodide;

8- (2'-pyridyl) -3 -methy1-G-methylthiopurine;

8- 2-pyridyl -6-thiopurine;

8- 2-pyridyl -3-methyl- 6-thiopurine;

8-(4-pyridyl)-6-thiopurine; and

8- (4-pyridyl) -6-methylthio purine.

The compounds of the invention are represented by the followingformulae:

N H-N The novel hypoxanthines, Xanthines and 6-thioxanthines of thepresent invention are obtained by direct condensation of the appropriateknown 4,5-diaminopyrimidines with an amidineI To form theG-merapfopurine compounds, sulphuration of the correspondinghypoxanthines gives better results than the condensation of 4,5-diamino-6-mercaptopyrimidine with an amidine. The pyridyl purines No. 4 and 5 inTable 1 can be prepared by dethiation of the 6-mercaptopurines Nos. 9and 10. However, the analogous reaction fails With8-(2'-pyridyl)-6-mercaptopurine (No. 8) or its methylthio ether and*8-(2'-pyridyl) purine (No. 3) can only be obtained by condensation of4,5-diaminopyrimidine with a salt of Z-amidinopyridine.

The N-methylpyridinium salts, listed in column (c) of Table 2, wereobtained by two methods: (a) by treatment of the novel tert.pyridylpurines No. 21, 22 (hypoxanthines), 26 and 27 (Xanthines) withmethyl iodide in dimethylformamide (DMF); and (b) by condensation of4,5-diaminopyrimidines with a quaternary amidiuium salt, as exemplifiedin the following scheme for 4-pyridyl derivatives:

from 1 to 5 is absent; otherwise the shape of the curves resembles thoseof FIGS. 1 and 2. The quaternary ions, when bearing no other charge,exhibit an absorption maximum similar to that of the protonated form ofthe corresponding tert. pyridyl derivatives. E.g. compound No. 22aabsorbs at 342 me, while the corresponding protonated form of No. 22shows its maximum at 336 me and the uncharged molecule of No. 19 at 330III/L. On the other hand, the quaternary 3-pyridyl derivative (No. 21a)absorbs at 305 me and the protonated form of No. 21 at 303 me (see Table2).

TABLE 1.PHYSICAL PROPERTIES OF S'SUBSTITUTED PURINES wnnx (mi Substit-RF in solvent uent at Uncharged Pyrldinium Akin. No. C-8 molecule (a)cation (b) (I)a) A B Fluorescence 1 I. Derivatives of purine 1 H 263 2Phenyl 207 30 Yellow. 3 2-pyridyl 299 308 9 70 73 17 D0. 4 B-pyridyl 296293 3 67 66 11 D0. 5 4-pyridyl 289 302 13 68 66 17 Do.

II. 6-mercapt0purines 6 H 7 Phenyl 65 Blue. 8 2-pyridyl 65 56 09Green-yellow. 9 3-pyridyl 54 44 09 Yellow-brown.

4-pyridyl 351 378 27 48 4O 1O Ycllowish.

III. G-thioxanthines 340 Phenyl 360 Orange. 2-pyridyl 365 Yellow.S-pyridyl 363 D0. 4-pyridyl 370 D0.

1 The colors refer to solvent A. TABLE 2.PHYSIOAL PROPERTIES OF8-SUBSTITUTED HYIOXANTHINES AND XAN'IHINES mnx.( I- of- M1. u) of"Uncharged Rm. i 01 Neutral Substituent at molecule Pyridinium A)\ H,N-methylpyri- AMMX. form 2 Mono-anion Bis-anion No. ()8 (a) cation (b)(bdinium ion 1 (c) (c-a) (c) (d) (c-a) Md-a) I. Hypoxanthines 1 Thesevalues refer to the form, in which the molecule bears no other chargeexcept at the quaternary nitrogen of the pyridine ring.

2 In the quaternary N-methylpyridinium derivatives 21(a) etc., thevalues in column (a) refer to the form hearing no other charge except atthe pyridine nitrogen.

lihis derivative does not form a bis-anion even when the pH is raised F}compound 21 the increase of km. still continues at pH 14 (see TABLE 3 D1* D 2* pKa {3K4 RF in solvent bismonomonobis- Fluores- No. Snbstituentat 0-8 cation cation anion anion A B O ceuce** I. Hypoxanthines 17Phenyl 1. 3 8. 3 12. 5 71 65 28 Blue. 18 p-Anisyl 0.5 9. 4 54 D0. 19p-Nitrophenyl 0. 8 7. 5 12. 0 40 Orarxgere 20 2-pyridyl 0. 6 1. 8 8. 512. 4 63 48 11 Greenish. 21 3-pyr1d 2 2. a 7. 7 1s .57 .38 .03 Blue21(a) Nimethylpyrldinium deriva- 0. 1 5. 5 11. 7 33 15 01 Yellow.

we. 22 4-pyridyl 0 3.4 7.0 12.4 .50 .33 .02 Blue. 22(a)Ngmethylpyridmium derlva- 0. 5 5. 5 11.4 33 14 01 Do.

II. Xanthines 24 Phenyl 0.6 7. 2 11.6 53 .44 28 D0. 25 2-pyr1dyl -1 1. 57. 0 l1. 6 50 39 09 Greenb no. 26 3-pyridyl -2 2. 5 6.8 11.7 .45 03 Do.26(a) Nirnethylpyridirnum deriva- -1 5. 4 10. 4 30 12 01 Bluish.

we. 27 it-pyridyl 2 3. 5 6. 6 10. 9 42 18 02 Greenblue. -0. 5 4. 4 10. 428 09 01 Blue.

27(a) N-methylpyridinium derivative.

*The pK1 values characterize the binding of two protons in the tort.pyridyl derivatives and of a single proton in all other purines listed.The pK values refer to the form, in which only the pyridine nitrogen hasE E E A B a A CHART 3 Absorption maxima of 8-(N-n1ethylpyridinium)'hypoxanthines as function of pH. e--o, 4-pyridy1 derivative (No. 22a;left-hand ordinate);

ea, 3-pyridyl derivative (No. 21a; right hand ordinate) I s 360 2 31s EE A 311. A 303 CHART 4. Absorption maxima of 8-(N-methylpyridinium)Xanthines as function of pH.

e-- e, 4-pyridyl derivative (No. 272; left-hand ordinate); ---'o,a-pyridyl derivative (No; 26a; right-hand ordinate).

ILLUSTRATIVE EXAMPLES Ultraviolet absorption spectra were determined ona Beckman DU spectrophotometer. For measuring changes of A as functionof pH, the following buffers were employed: pH 3, 27 NH SO 1, 11NH SO pH03, HClO 4 and 5, 0.1 M acetate buffer; pH 6-8, 0.1 M phosphate buffer;9 and 10, 0.1 M borate buffer; pH 11-14, NaOH. Paper chromatograms weredeveloped on Whatman paper No. 1 by the descending method, using thefollowing solvents: A (neutral solvent), 95% EtOH-dimethylformamide-H O3:1:1 (v./v.); B, n-butanol- AcOH-H O 12:3:5 (v./v.); C (stronglyacidic), 95% 'EtOH-AcOH-5% HCl 17: 1:1 (v./v.). Spots were detected 8 bytheir fluorescence under a Mineralight ultraviolet lamp 255 mp).

Preparation of amidines 2-amidinopyridine hydrochloride (F. C. Schaeferand G. A. Peters, J. Org. Chem., 1961, 26, 412), the corresponding3-amidino derivative (H. J. Barber and R. Slack, J. Amer. Chem. Soc.,1944, 66, 1607) and the 4-isomer (F.C. Schaefer and G. A. Peters, J.Org. Chem., 1961, 26, 412; T. S. Gardner, E. Wenis and J. Lee, J. Org.Chem., 1954, 19, 753) have been described in the literature. We havefound that all three isomers can be conveniently prepared by the methodof Schaefer and Peters (F. C. Schaefer and G. A. Peters, J. Org. Chem.,1961, 26, 412) in yields of 70-75%. 1-methyl-4-amidinopyridinium saltwas synthesized according to Poziomek (E. J. Poziomek, J. Org. Chem.,1963, 28, 590).

1-methyl-3-amidinopyridinium iodide hydriodide was obtained as follows:A solution of 3-amidinopyridine hydrochloride (15 g.) in DMF (150 ml.)and methyl iodide (30 ml.) was kept at room temperature for 24 hr. Thesolvent was removed in vacuo and the residue recrystallized frommethanolethyl acetate (4:1, v./v.). Yellowish needles (30 g., M.P. 214;A (PH 7) 225 and 262 m (Found (percent): C, 22.1; H, 2.9; N, 10.7; I,64.6. C H N I requires (percent): C, 21.5; H, 2.8; N, 10.7; I, 65.0).

p-Anisamidine hydrochloride was prepared according to Fanta and Hedman(P. F. Fanta and E. A. Hedman, J. Amer. Chem. Soc., 1956, 78, 1434) andp-nitrobenzamidine by the method of Andrews et al. (C. H. Andrews, H.King and J. Walerr, Proc. Roy. Soc. (London), 1946, B 133, 20). Thepyrimidines were all synthesized according to the literature:4,5-diaminopyrimidine (D. J. Brown,

' J. Appl. Chem., 1952, 2, 239); its 6-hydroxy derivative (A. Albert, D.J. Brown and G. Cheeseman, J. Chem. Soc., 1951, 474); 4,5-diaminouracil(M. T. Bogert and D. Davidson, J. Amer. Chem. Soc., 1933, 55, 1668);4,5- diamino-6-thiouracil (G. Levin, A. Kalmus and F. Bergmann, J. Org.Chem., 1960, 25, 1752); 4,5-diamino-6- hydroxy-3-methylpyrimidine (Brownand Jacobsen, J. Chem. Soc. 1965, p. 1175) and the l-methyl derivativethereof (Elion, J. Org. Chem., 1962, 27, 2478).

8-substituted purines General procedure.The appropriate4,5-diaminopyrimidine, usually as free base, was thoroughly mixed in amortar with two equivalents each of anhydrous sodium acetate and theamidine salt. The temperature of the mixture was slowly raised untilevolution of ammonia set in, and heating was continued until the meltresolidified. The cake was then treated with 1 N-NaOH and the mixturedecolorized with charcoal and filtered. The hot filtrate was neutralizedwith glacial acetic acid or by addition of solid ammonium chloride. Thespecial conditions required for isolation of each product and theanalytical results are summarized in Table 4.

EXAMPLE 1 6'-mercapto-S-phenylpurine (No. 7)

A mixture of 8-phenylhypoxanthine (No. 17), (2.2 g.), phosphorouspentasulphide (10 g.) and dry pyridine ml.) was stirred and refluxed for3.5 hr. The solvent was removed in vacuo, and the residue treated withcold Water (40 ml.) for 30 min. and subsequently heated to 70 for 60min. The insoluble portion was taken up in 1 N-NaOH, the mixture warmedand treated with charcoal and the filtered solution acidified withglacial acetic acid. Recrystallization from dil. acetic acid gavelightbrown branched needles (2.2 g., 93%), decomposing at 330 (Found(percent): C, 57.2; H, 3.6; N, 24.2; C H N S requires (percent): C,57.9; H, 3.5; N, 24.6).

EXAMPLE 2 8-phenyl-6-thioxanthine (No. 12)

This compound was obtained by sulphuration of 8- phenylxanthine (No.24), using the procedure just described. Branched needles, (93%),decomposing at 330 (Found (percent): C, 52.8; H, 3.5; N, 22.5. C H N OS-0.5 H 0 requires (percent): C, 52.2; H, 3.6; N, 22.1).

The novel 6-thioxanthines (No. 13, 14 and 15) and the novelhypoxanthines (N0. 18, 19, 20, 21 and 22) were obtained by directcondensation of the appropriate known 4,5-diaminopyrimidines with anamidine following the process of Bergmann and T amari. (J. Chem. Soc.,1961, 4468).

EXAMPLE 3 8-(2-pyridyl)hypoxanthine (No. 20)

An intimate mixture of 5 grams of 4,5-diamino-6- hydroxypyrimidine (freebase), grams of 2-amidinopyridine hydrochloride and 5 grams anhydroussodium acetate was heated to 160l80 for 20 minutes. The cake was treatedwith 1 N-NaOH; the mixture was boiled in the presence of active charcoaland filtered. The filtrate was neutralized by the addition of solidammonium chloride. Recrystallization from DMFH O=1:1 gave colorlessneedles of dec. p. -3l0. Yield 83% A (pH 5) 299 m (pH 10) 307 mi.

Analysis.Calcd. for C H N O (percent): C, 56.3; H, 3.3; N, 32.9. Pound(percent): C, 56.5; H, 3.1; N, 33.1.

EXAMPLE 4 8-(3'-pyridyl)hypoxanthine (No. 21)

An intimate mixture of 4,5-diamino-6-hydroxypyrimidine (free base) (5g.), 3-amidinopyridine hydrochloride (10 g.) and anhydrous sodiumacetate (5 g.) was heated to 190 for minutes. The cake was treated with1 N-NaOH and the mixture boiled with charcoal and filtered. The filtratewas neutralized by addition of solid NH Cl. Purification was effected bythe same procedure as above. The product, obtained in 81% yield, formscolorless needles, dec. p. 310; A (pH 6) 290 mu; (pH 1) 302 my; (pH 11)303 mu.

Analysis.Calcd. for C H N O (percent): C, 56.3; H, 3.3; N, 32.9. Found(percent): C. 55.8; H, 3.5; N, 32.6.

EXAMPLE 5 8-(4-pyridyl)hypoxanthine (No. 22)

An intimate mixture of 4,S-diamino-6-hydroxypyrimidine (free base) (5g.), 4-amidinopyridine hydrochloride (10 g.) and anhydrous sodiumacetate (5 g.) was heated in a metal bath to 180200 during 5 minutes.The cake was treated with 1 N-NaOH and the mixture then boiled brieflyin the presence of active charcoal. The mixture was filtered and to thefiltrate solid ammonium chloride was added, until the pH declined toabout 7.5. The precipitate was separated after cooling and purified bydissolution in 1 N-NaOH and was neutralized with ammonium chloride.Colorless needles of dec. p. -310; yield 70% A (pH 9) 311 mu, (pH 6) 295m Analysis.-Calcd. for C H N O (percent): C, 56.3; H, 3.3; N, 32.9.Found (percent): C, 55.9; H, 3.5; N, 32.9.

EXAMPLE 6 8-(2-pyridyl)-6-thioxanthine (No. 13)

An intimate mixture of 4,5-diamino-6-thiouracil (free base) (5 g.),2-amidinopyridine hydrochloride (10 g.) and anhydrous sodium acetate (5g.) was heated to 180-190 for 10 minutes. The cake was treated with 1N-NaOI-I- and the mixture boiled in the presence of active charcoal andfiltered. The filtrate was neutralized with solid ammonium chloride andcooled. The precipitate crystallized from DMF-H O=1:1 in yellow 10plates of dec. p. -300, yield 45%. )x (pH 8) 371 mu; (pH 1.0) 391 mu.

Analysis.Calcd. for C H N OS (percent): C, 49.0; H, 2.9; N, 28.6; S,13.1. Found (percent): C, 49.0; H, 3.4; N, 28.6; S, 13.4

EXAMPLE 7 8-(3'-pyridyl)-6-thioxanthine (No. 14)

An intimate mixture of 4,5-diamino-6-thiouracil (free base) (5 g.),3-amidinopyridine hydrochloride (10 g.) and anhydrous sodium acetate (5g.) was heated for 15 minutes to 190. The cake was dissolved in 1N-NaOI-I, the mixture boiled with active charcoal and filtered. Thefiltrate was neutralized by addition of solid ammonium chloride.Purification by the same method gave yellow prisms of dec. p. -310,yield 94%; A (pH 8) 363 mu.

Analysis.Calcd. for C10H7N5OS0.5 H O (percent): C, 47.2; H, 3.1; N,27.6; S, 12.6. Found (percent): C, 46.6; H, 2.7; N, 27.4; S, 12.7

EXAMPLE 8 8-(4-pyridyl)-6-thioxanthine (No. 15)

An intimate mixture of 4,5-diamino-6-thiouracil (free base) (5 g.),4-amidinopyridine hydrochloride (10 g.) and anhydrous sodium acetate (5g.) was heated to 260 for 20 minutes. The cake was treated as before.Purification in the system 1 N-NaOHNH Cl gave yellow needles of dec. p.-310. Yield 64%; k (pH 8.0) 372 my; (pH 1) 397 mu.

Analysis.Calcd. for C H O NS-0.25 H O (percent): C, 48.1; H, 3.0; S,12.8. Found (percent): C, 48.0; H, 3.4; S, 12.8.

EXAMPLE 9 8-(2-pyridyl)-6-mercaptopurine (No. 8)

Thiation of the appropriate hypoxanthine (No. 20) was carried out inboiling B-picoline. From dilute acetic acid yellowish needlesdecomposing at 300 (Found (percent): C,52.1; H, 3.1; N, 30.9; S, 13.9. CH N S requires (percent): C, 52.4; H, 3.1; -N, 30.6; S, 13.9).

EXAMPLE 1O 6-methylthio-8- (2'-pyridyl) purine The 6-mercapto derivativeNo. 8 (1 g.) in 1 N-NaOH (25 ml.) was stirred with metyl iodide (1.7 g.)at room temperature for 2 hr. Acidification with glacial acetic acidcaused precipitation of the thio ether. From dil. acetic acid whiteneedles (0.43 g., 45%), decomposing at 300; A (pH 8) 241 and 326 mu(Found (percent): C, 54.2; H, 3.6; N, 29.2; S, 13.2. C H N S requires(percent): C, 54.3; H, 3.7; N, 28.8; S, 13.2).

EXAMPLE 11 8- (3 -pyridyl) -6-mercaptopurine (No. 9)

This compound was prepared from the hypoxanthine 21 by the proceduredescribed for compound No. 8. From dilute acetic acid yellow prisms(68%) decomposing at 310 (Found (percent): C, 52.4; H, 3.2; N, 30.8; S,13.8).

EXAMPLE 12 8- (4-pyridyl) -6-mercaptopurine (No. 10)

This product was prepared as described directly above for the isomers.For purification, it was dissolved in 1 N-NaOH and precipitated byaddition of solid ammonium chloride. Yellow needles (82%), decomposingat 300 (Found (percent): C, 52.0; H, 3.4; N, 30.3; S, 13.5).

EXAMPLE 13 8-(3'-pyridyl)purine (N0. 4)

A solution of 6-mercapto 8-( 3'-pyridyl) purine (No. 9) (4 g.) in 1N-NaOH (400 ml.) was refluxed for 3.5 hr.

in the presence of Raney nickel (28 g.). After the catalyst had beenremoved by filtration, the solution was neutralized and brought todryness in vacuo. The residue was extracted with concentrated ammonia at80, the mixture filtered and the filtrate brought to pH 6.5 by additionof acetic acid. Upon cooling overnight, 1.6 g. (34%) of white crystalsprecipitated. Recrystallization from water gave colorless needlesdecomposing at 300. Found (percent): C, 61.1; H, 3.9; N, 35.4. C H Nrequires (percent): C, 60.9; H, 3.6; N, 35.5).

EXAMPLE 14 8-(4'-pyridyl)purine (No. 5)

Prepared as above by desulphurization of the 6-meicaptopurine No. 10.From water colorless needles (80% decomposing at 300 (Found (percent):C, 61.0; H, 3.4; N, 35.7). No. 3 was prepared in the same manner fromthe 6-mercaptopurine No. 8.

EXAMPLE 1 5 Xanthines (Nos. 25, 26 and 27) The 2-pyridyl (No. 25), the3-pyridyl (No. 26) and the 4-pyridyl (No. 26) Xanthines were prepared bycondensation of 4,5-diaminouracil with the proper amidine salt andanhydrous sodium acetate under the conditions set forth in Table 4.

EXAMPLE 16 Preparation of 8-(1'-methylpyridinium)purines iodides orbetaines 8-(3'-pyridyl)hypoxanthine 1-methiodide (No. 21a) (a) Asuspension of compound No. 21 (1.3 g.) in DMF (120 ml.) and water (60ml.) was stirred with methyl iodide (20 ml.) at 50 for 20 hrs. Thesolution, which had become homogeneous, was brought to dryness in vacuoand the residue was crystallized from 50% isopropanol. Bushels ofneedles (1.2 g., 55%), MP. 248- 2 50 (Found (percent): C, 37.1; H, 3.2;N, 19.5. C H N OI requires (percent): C, 37.2; H, 2.8; N, 19.7). (b) Amixture of 4,S-diamino-6-hydroxypyrimidine (A. Albert, D. J. Brown andG. Cheeseman, J. Chem. Soc., 1951, 474) (1.3 g.) and1-methyl-3-amidino-pyridinium iodide hydriodide (3 g.) was heated to 170for 5 min. The resulting black tar was extracted with hot water and themixture filtered. From the filtrate, the condensation product wasprecipitated by addition of isopropanolethyl acetate (1:1, v./v.).Crystallization from 50% isopropanol gave 0.3 g. (11%) of colorlessneedles, identical in every respect with the product obtained byprocedure (a).

EXAMPLE l7 8-(3'-N-methylpyridinium)xanthine betaine (No. 26a) (a)Methylation of the xanthine 26 was carried out in 80% DMF at 50 during 6hr. The quaternary salt (65% yield) settled directly upon cooling at thereaction mixture. The salt was dissolved in warm water and ammonia addedto the solution. The betaine of 26a crystallized in colorless needles,decomposing at 310. (Found (percent): C, 52.2; H, 4.3; N, 28.1. C H N O-0a5 H O requires (percent): C, 52.4; H, 4.0; N, 27.8). The picrate ofNo. 26a formed yellow prisims for water, M.P. 314. (Found (percent): C,43.1; H, 3.0; N, 24.2. C H N O requires (percent): C, 43.2; H, 2.5; N,23.7%). (b) An intimate mixture of 4,5-diaminouraci1 (M.T. Bogert and D.Davidson, J. Amer. Chem. Soc., 1933, 55, 1668) (1.2 g.),1-methyl-3-amidinopyridinium iodide hydriodide (3 g.) and anhydroussodium acetate (2 g.) Was heated to 220 for min. The cake was extractedwith hot water and the mixture filtered. Upon cooling the filtrate, thecondensation product (20%) precipitated as iodide. It was converted intothe betaine, which proved to be identical with the compound obtained byprocedure (a).

EXAMPLE 18 8-(4'-N-methylpyridinium)hypoxanthine betaine (N0. 22a) (a) Asuspension of the hypoxanthine 22 (2 g.) in 70% DMF (200 ml.) and methyliodide (20 ml.) was stirred at for 5 hr. The reaction mixture, which hadbecome homogeneous, was brought to dryness and the residue dissolved indil. ammonia. After decolorization with charcoal, filtration and coolingthe betaine (1.5 g., 70%) crystallized in yellow, elongated plates,which were however difficult to purify. For analysis, the picrate provedsatisfactory. Yellow plates, decomposing -300 (Found (percent): C, 44.6;H, 2.7; N, 24.5. C H N O requires (percent): C, 44.7; H, 2.6; N, 24.6).(b) A mixture of 4,5-diamino-6-hydroxypyrimidine (A. Albert, D. J. Brownand G. Cheeseman, J. Chem. Soc., 1951, 474) (4.5 g.),1-methyl-4-amidopyridinium iodide hydriodide (4 g.) and sodium acetate(2 g.) was heated to 205 for 10 min. The cake was extracted with hotammonia, the solution decolorized with charcoal and the filtrate cooled.The betaine of 22a was obtained in 30% yield. Its picrate was identicalwith the one described under (a).

EXAMPLE 19 8-(4'-N-methylpyridinium)xanthine betaine (No. 27a) (a)Condensation of 4,5-diaminouracil with the appropriate amidine wascarried out at 210 during 25 min. The betaine, obtained as before,crystallized from water in hairlike needles of MP. 300; yield 30% (Found(percent): C, 50.0; H, 4.4: N, 26.0. C H N O -H O requires (percent): C,50.6; H, 4.2; N, 26.7). (b) A suspension of the xanthine 27 (2 g.) in70% DMF (200 ml.) and methyl iodide (50 ml.) was refluxed for 16 hr. Themixture was filtered and the filtrate brought to dryness. The residuecrystallized upon trituration with ethyl acetate. Yield 1.6 g., 50%. Theproduct was identical in all properties with the compound, resultingfrom procedure (a).

EXAMPLE 20 3-methyl-8 (2'-pyridyl)hypoxanthine An intimate mixture of4,5-diamino-6-hydroxy-3- methyl-pyrimidine (14 g.) and Z-amidinopyridinehydrochloride (28 g.) was heated to 180 for 30 min. The cake wasextracted with boiling methanol, the liquid decanted,

decolorised with charcoal and filtered. The substance crystallised incolorless rods of dec. p. 300; yield 48%.

X (pH 8) 314 mu; R (solvent A) 0.62 Analysis.'Calcd. for C H N O(percent): C, 58.1; H, 3.9; N, 30.8. Found (percent): C, 57.7; H, 4.2;N, 31.2.

EXAMPLE 21 3-methyl-6-methylthio-8 (4'-N-methylpyridinium) purine iodide8(4-pyridyl)-6-mercaptopurine (Example 12) (10 g.) was suspended in DMF(1 liter). The mixture was stirred and refluxed with methyl iodide (60ml.) for 2.5 hr. The solvent was removed in vacuo and the residuetriturated' with ethanol. The solid material (9.5 g. 54%) crystallisedfrom water in yellow needles, dec. 280.

Analysis.Calcd. for C H N SI (percent): C, 29.1; H, 3.5; N, 17.5; S,8.0. Found (percent): C, 39.2; H,

A (pH 8) 278 and 369 m R (A) 0.44

EXAMPLE 22 3-methyl-8 (4-N-methylpyridinium -6thiopurine betaine Into asolution of ammonia (200 ml.), that was saturated at 0 with gaseoushydrogen sulfide, 3-methyl-6- methylthio-8(4-N-methylpyridinium) purineidodide (Ex- 13 ample 21) (20 g.) was added in small portions. Themixture was kept at and H 8 was bubbled through for. a further 30 min.The precipitate crystallised from water in reddish needles, dec. p. 200;yield, 30%.

h (pH 8) 430 mg; R (A) 0.67

Analysis.Calcd. for C H N S-2H O (percent): C, 49.1; H, 5.1; N, 23.9; S,Found (percent): C, 49.1; H, 5.1; N, 23.9; S, 10.9.

EXAMPLE 23 1-methyl-8(4'-pyridyl) hypoxanthine An intimate mixture of4,5-diamino-6-hydroxy-l-methylpyrimidine g.), 4-amidinopyridinehydrochloride (60 g.) and anhydrous sodium acetate (20 g.) was heated to220-240 for 15 min. The cake was extracted with 10% ammonia, the mixturefiltered and the filtrate acidified with acetic acid. Yellow needles ofdec. p. 300, yield: 60%.

A (pH 8) 312-313 mg; R (A) 0.63 Analysis.-Calcd. for C H N O (percent):C, 58.1; H, 4.0; N, 30.8. Found (percent): C, 58.0; H, 4.0; N, 31.0.

EXAMPLE 24 1-methyl-8 (4'-pyridyl )-6-thiopurine A suspension of1-methyl-8(4-pyridyl) hypoxanthine (see Example 23) (24 g.) and ofphosphorous pentasulfide (72 g.) in ,B-picoline (1 liter) was stirredand refluxed for 2 hr. The solvent was distilled off, the residuetreated with warm Water and filtered. The solid portion was dissolved inlN-NaOH, the solution decolorised with charcoal and filtered. The sodiumsalt of the desired product crystallised upon cooling. It was removed byfiltration, dissolved in hot water and the thiopurine precipitated asfree base by addition of ammonium chloride.

l-methyl-8(4-pyridyl)-6-thiopurine crystallises in yellow needles, dec.p. 300, yield, 40%.

A (pH 8) 262; 352353 III 1.; R (A) 0.67

Analysis.--Calcd. for C H N S (percent): C, 54.3; H, 3.7; N, 28.8; S,13.2. Found (percent): C, 54.2; H, 4.0; N, 29.0; S, 13.1.

EXAMPLE 1-methyl-6-methylthio-8(4'-pyridyl) purine A solution of thethio compound, described in Example 24 (10 g.) in 0.5 N-NaOH (120 ml.)was stirred at room temperature with methyl iodide (3.2 ml.) for 90 min.The precipitate was removed and recrystallised from water. Yellowishneedles, dec. 190; yield, 68%.

A (pH 8) 335 mp; R (A) 0.64-

Analysz's.Calcd. for C H N S (percent): C, 56.0; H, 4.2; N, 27.2. Found(percent): C, 55.4; H, 4.6; N, 27.7.

EXAMPLE 26 1-methyl-6-methylthio-8 (4'-methylpyridinium) purine iodide Asolution of the thioether, described in Example 25, (10 g.) in DMF (600ml.) and methyl iodide ml.) was stirred at room temperature for 2 hrs.The solid that had separated was recrystallised from 90% isopropanolYellowish needles, M.P. 245; yield quantitative.

i (pH 8 276, 361 mu; R (A) 0.32 Analysis.-Calcd. for C H N SI (percent):C, 39.1; H, 3.5; N, 17.5; S, 8.0. Found (percent): C, 39.1; H, 3.8; N,17.6; S, 7.7.

EXAMPLE 27 3-methyl-6-methylthio-8 (2'-pyridyl) purine A suspension of8(2'-pyridyl)-6-mercaptopurine (Example 9) (25 g.) in DMF (600 ml.) andmethyl iodide 14 (50 ml.) was stirred and refluxed for 1 hr. The solventwas removed in vacuo, the residue dissolved in warm water and the pH wasadjusted to 10 by addition of ammonia to precipitate the methylthioether as free base. From water, white needles, M.P. 252; yield, 32%.

h (pH 8) 2.43, 340 my; R (A) 0.67

Analysis.Calcd. for C H N S (percent): C, 56.0; H, 4.3; N, 27.2; S,12.5. Found (percent): C, 56.5; H, 4.3; N, 27.2; S, 12.6.

(II) Biological activities.The compounds described in the foregoingexamples were tested for biological activity and showed results asexemplified by the following:

(1) Marked and prolonged hypotension was produced by intravenousinjection into cats, which were anesthetized with nembutal, of

8-pyridylxanthines 3-methyl-6-methylthio-8 (2-pyridyl) purine (Example3-methyl-8 (4-N-methylpyridinium) -6-thiopurine betaine (Ex. 19a)3-methyl-6-methylthio-8 (4'-N-methylpyridinium) purine iodide, (EX. 19)

(2) The 8-pyridyl-6-thiopurines and their S-methyl ethers showed markedinhibition of E0 771 mammary adenocarcinoma, S-180 sarcoma and Ehrlichascites carcinoma in mice-as follows:

BR-l 8- 2'-pyridyl) -6-thiopurine BR-2 8- 2'-pyridyl)-6-methylthiopurine BR-3 8- 2-pyridyl) -3-methyl-6-thiopurine BR4= 8-(2'-pyridyl) -3 -rnethyl-6-methylthiopurine BR8= 8- (4-pyridy1)-6-thiopurine BR9 8- (4'-pyridyl) -6-methylthiopurine The compounds werereceived as crystalline substances with known formulas. They were notsoluble in the concentrations suitable for tumor studies, therefore theywere homogenized by using a homogenizer of the Potter- Elevehjem type;the suspending agent used was 0.5% carboxymethyl cellulose in saline.All materials were injected intraperitoneally in mice at a volume notexceeding 0.5 ml. per injection.

Tumors.The experimental mouse tumors used were E0771 mammaryadenocarcinoma, Sarcoma 180 (S-180) and Ehrlich ascites carcinoma(Lettre).

The solid tumors E0771 and S-180 were transplanted by the usual asepticprocedures using the trocar method. Implantation of Ehrlich ascitescarcinoma was performed by i.p. injection of 2 10 tumor cells per mouse.

Hosts-S-l and Ehrlich ascites carcinoma were grown in random bred Swissmice and E0771 in C57BL inbred mice.

Chemotherapy.All compounds were injected intraperitoneally 24 hoursafter tumor implantation. Injections were repeated every 24 hours. Thenumber of days of treatment, however, varied with the test system(Tables 1, 2, 3). All compounds were tested using groups of 6-8 mice foreach type of tumor. Death and body weight changes in the treated andcontrol groups were obtained and recorded (Tables 5, 6 7). The animalswere sacrificed one day after the last injection for the evaluation ofthe carcinostatic efiect. The tumor volume (TV) as well as the totalpacked cell volume (TPCV) for the Ehrlich ascites carcinoma and thecorresponding tumor weight for S- and E0771 were established accordingto Cancer Chemotherapy National Service Center of United States (CCNSC)specifications (1959).

The results are given as percent of tumor growth inhibition as comparedto tumor growth on the control animals.

Results-Results obtained are shown in Tables 5, 6 and 7. Experimentswere repeated whenever a positive response was obtained (50% inhibitionof tumor growth 15 16 was considered as a positive result; the samecriteria is El S used by CCNSC of USA). I N H N Analysis of Table showsthat the Ehrlich ascites carcinoma tumor was not sensitive to thesecompounds. CH3 N CH3 I The other two systems of tumors used, namelyS-180 5 and E0771, showed a positive response to all the com- N N Npounds tested. The highest percentages of tumor growth inhibition (Table6) were obtained in the B0771 system with compounds BR2 and BR8 whichwere used in repeated experiments.

R1 S 7 Administration of BR8 in ,s 1s0 resulted in the high- N estanti-tumor activity (Table 7). BR9 showed a posii J and m H N N H N N lH tive anti-tumor activity in the S-180 tumor system.

Thus, of the six compounds submitted for anti-tumor I screening, allshowed positive anti-tumor eifects in the CH3 CH3 Sarcoma 180 (S180) andmouse mammary adenocar- (5) (6) cinoma E0771. BR-2 and BR8 proved to bethe most active with respect to tumor inhibition. Tumor growth inwhere Ris -SCH SH, or SCH I; and R =pyridy1 hibitions of up to 89% wereobtained by these compounds. or N-methylpyridinium.

TABLE 5 Tumor response Percent inhi- Animal bitlon of tumor Mortalityweight growth Daily dose, Route of Number of number/ change TumorCompound rug/kg. injection treatments total gr. T/' TV TPCV BR-l 100I.p. 6 0/6 32 BRZ 500 I.p. 6 6 46 47 BR3 500 I.p. 6 0/6 32 29 BR4 500I.p. 6 0/6 17 25 BR8 50 I.p. 6 3/6 11 0 BRQ 100 I.p. 6 0/6 67 69 BR9 100I.p. 6 1/6 29 62 TABLE 6 Tumor response Animal Percent Mortality weightinhibition Daily dose, Route of Number of number/ change of tumor TumorCompound mgJkg. injection treatments total gr. T/d growth E0771 BR-l 100I.p 11 0/6 -2 64 E0771 BR-l 100 I.p 10 o a +2 42 E0771 BR-z 500 I.p 110/6 -2 s9 E0771 BR2 100 I.p 11 0/6 0 75 E0771 BR-3 500 I.p 11 0/6 0 69E0771 BR4 500 I.p 11 0/6 1 61 E0771 BIZ-4 100 I.p 10 0/6 +2 59 E0771 BR8I.p 11 2/6 5 88 E0771 BR8 50 I.p 10 1/6 0 71 TABLE 7 Tumor responseAnimal Percent Mortality weight inhibition Daily dose, Route of Numberof number/ change, of tumo Tumor Compound mg./kg. injection treatmentstotal gr. T/C' growth 100 I.p. 11 0/8 2 08 100 I.p. 10 0/8 +2 61 500I.p. 11 0/8 --4 61 500 I.p. 10 8 +1 68 500 I.p. 11 8 -1 500 I. 11 0/8 -139 50 I.p. 11 0/8 -3 74 50 I.p. 10 2/8 3 100 I.p. 11 0/8 0 61 What isclaimed is: 2. A compound according to claim 1, consisting of 1. Acompound selected from the group consisting of8-(2'-pyridyl)-6-methylthio purine. the following formulae: 3. Acompound according to claim 1, consisting of 8- (4'-N-methy1 pyridinium)-3-methyl-6-methylthio purine R1 8 iodide II A N N 4. A compoundaccordmg to claim 1, consisting of N R HN8-(4'-pyridyl)-1-methyl-6-thiopurine. H I N I 5. A compound according toclaim 1, consisting of N 8- (4-pyr1dy1)-1-methyl-6-methylthiopurine.

I 6. A compound according to claim 1, consisting of8-(4-N-methylpyridinium)-1-methyl-6-methy1thio purine (1) 75 iodide.

17 18 7. A compound according to claim 1, consisting of 14. A compoundaccording to claim 1, consisting of8-(2'-pyridyl)-3-methyl-6-methylthiopurine. 8-(4-pyridyl)-6 mercaptopurine.

8. A compound according to claim 1, consisting of 8-(2'-pyridyl)-6-thiopurine. References Cited 9. A compound according to claim 1, consistingof 5 UNITED STATES PATENTS 8- 2'-pyridyl) -3-methyl-6-thiopurine.

10. A compound according to claim 1 consisting of f g8-(4'-pyridy1)-6-thiopurine. 11c er et a 11. A compound according toclaim 1 consisting of ALEX M AZEL Primary Examiner8-(4'-pyridyl)-6-methylthio purine. 10

12. A compound according to claim 1 consisting of TIGHE, Assistant mmer8-(2'-pyridy1)-6 mercapto purine.

13. A compound according to claim 1 consisting of 8-(3-pyridyl)-6mercapto purine. 260-216, 253; 424-253 UNITED STATES PATENT OFFICECERTIFICATE OF CORRECTION Patent No. '3 573:3Q9 Dated M h 3Q 192 Inventr( Felix Gotthi 1f BERGMANN et a1.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 2, line 38, after the group of formulas, insert where R SCH -SH,or SCH I R pyridyl or N-methylpyridinium.-

Column h, Table 2, No. 23" under the heading "Bis-anion (d) delete "383"and insert ---283--.

Column 5, Table A, No. 3, under the heading "Formula" delete "C H N 'O.5H 0" and insert Column 15, Claim 1, formula (2), delete this formula asit appears and insert the following therefor:

Signed and sealed this Zhth day of August 1971.

(SEAL) Attest:

EDJARD M.FLETCHER,JR. WILLIAM E. SCHUYLER, JR. Attesting OfficerCommissioner of Patents

